Mycobacterium avium has emerged as a major opportunistic pathogen in AIDS patients. M.avium normally resides within the vacuolar compartment of the macrophage where it restricts access to the digestive and processing capabilities of the host cell. Access appears to be restricted by impaired membrane protein trafficking into and out of the M.avium and M.tuberculosis phagosome. Because of this and the similarity of intracellular M.avium to M.tuberculosis, M.avium may provide an ideal organism to investigate fundamental mechanism of protein traffic in mononuclear phagocytes. Newly formed phagosomes fuse and exchange membrane with multiple intracellular compartments including lysosomes and that the exchange of such proteins is critical to intracellular killing. We have developed methods to reconstitute phagosome fusion events in vitro and we have begun to identify the factors that are required for phagosome membrane traffic. Among the proteins that are likely to play structural and regulatory roles in phagosome membrane trafficking are the GTP binding proteins (Heterotrimeric G proteins, Rab GTP binding proteins and ADP- ribosylation factors-ARFs ) and the newly described SNARE docking proteins (i.e., receptors for the NEM-sensitive fusion protein ). Our initial experiments have successfully reconstituted the in vitro fusion of early phagosomes with endosomes. We propose to exploit the in vitro assay to determine the biochemical requirements for fusion of endosomes with phagosomes isolated from normal macrophages and from macrophages infected with M.avium. Our initial effort is to identify key GTP-binding proteins and SNARE docking proteins that mediate protein trafficking into and out of normal and M.avium phagosomes. Using membranes and cytosol from M.avium infected cells, we will investigate possible inhibitors produced by the organism. In our initial experiments we have begun to catalogue the biochemical changes occurring in phagosomal membranes during phagosome maturation. We propose to develop in vitro assays to reconstitute fusion of phagosomes with vesicles derived from the trans-Golgi Network and with lysosomes and to use these assays to determine whether products of M.avium intracellular infection impair membrane transport. Lastly, we will utilize in vitro assays using Mycobacterium avium to determine the influence of the live organism on the fusion of phagosomes with endosomes, vesicles derived from the trans Golgi Network and lysosomes.